Autoimmunity is the third most common category of disease in the United States, and its incidence is rising. Pemphigus vulgaris (PV) is a debilitating autoimmune disease characterized by blistering of the skin and mucous membranes. The disease is caused by serum autoantibodies (autoAbs) against desmoglein 3 (Dsg3), a protein that mediates keratinocyte cell adhesion. Currently, it is unknown why or how autoimmunity occurs in PV. A deeper understanding of how B cells become autoreactive to a peripheral antigen in a model human autoimmune disease such as PV, in which the disease autoantigen is well- characterized, is essential to understand the pathophysiology of disease and optimal treatment strategies. In order to differentiate into an antibody-secreting cell, all B cells must first randomly rearrange certain gene segments to express a receptor that can bind foreign antigens, a process called V(D)J recombination. Upon interacting with antigen, a B cell can acquire additional somatic mutations that increase affinity for antigen, thus maturing the immune response. However, these stochastic processes of B cell development are likely to produce B cells that bind self, requiring elimination of these autoreactive cells to prevent autoimmunity. In PV, a defect in B cell development occurs such that Dsg3-reactive B cells are not eliminated from the repertoire, resulting in the expression of anti-Dsg3 Abs and the development of disease. Our laboratory has previously shown that anti-Dsg3 Abs in PV patients use a limited number of variable region sequences, with shared variable region gene usage among different patients, implying common mechanisms of disease development. We have shown that a subset of these Abs bind Dsg3 even in the absence of somatic mutation, implying autoreactivity of the naive B cell repertoire. Intriguingly, these same variable region sequences have been described in the immune response to rotavirus, HIV, and influenza. The goals of the current research proposal are two-fold: 1) to identify pathologic B cell subsets in PV, defined as those that exhibit Dsg3-binding B cells in PV patients but not unaffected individuals; and 2) to determine whether Abs that recognize Dsg3 also bind foreign antigens, thereby explaining their persistence in the repertoire. Flow cytometry, single B cell PCR and ELISPOT utilizing patients' whole blood will reveal B cell subsets that are enriched for Dsg3-reactive B cells in comparison to unaffected individuals. Multiple approaches will be used to identify anti-Dsg3 Abs that are cross-reactive to foreign antigen, including directed immunochemical assays and high-throughput hybridoma and phage display screening techniques. Through the proposed research plan, we may validate novel therapeutic targets for PV that may more safely or effectively capture the disease-relevant B cell populations, via targeting of certain VH Ab families or more specific B cell subsets. Furthermore, we will gain a much better understanding of how autoAbs arise in PV, and therefore a deeper understanding of the pathogenesis of autoimmune disease as a whole.